Ishita Mukerji

Professor and Chair, Molecular Biology and Biochemistry Department
Ph.D. (Chemistry) University of California

imukerji@wesleyan.edu | 860-685-2422

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Protein and nucleic acid structure; structure-function relationships in protein-nucleic acid and protein-protein interactions using UV resonance Raman spectroscopy

We employ fluorescence and UV resonance Raman spectroscopic methods for probing protein-nucleic acid and protein-protein interactions. The Raman effect can be enhanced by several orders of magnitude by exciting into or near to an absorption band. Thus, the excitation wavelength can be used to probe different regions of the macromolecules. For example, an excitation wavelength of 230 nm selectively investigates the aromatic residues, Tyr and Trp; whereas, 260 nm selectively probes nucleic acid residues. We exploit the resonance effect to separately investigate DNA conformation from protein structure.

Protein-Nucleic Acid Interactions. Nucleotide-binding proteins play an extremely important role as regulators of genomic function. However, the molecular mechanism of these processes is not well understood, since only a few crystal structures exist for protein-nucleic acid complexes. We are addressing the mechanism of protein-mediated regulation of genetic processes, such as repression, recombination or expression by investigating the nucleotide-protein interface for a class of prokaryotic his tone-like proteins. The stabilization of DNA in coil or loop structures is the postulated mechanism by which these proteins participate in replication and inversion reactions and also enhance binding of proteins such as Lac repressor and camp-activator protein. It is this protein-induced deformation of DNA structure, which in turn modulates its genetic function, that motivates our investigations.

We are studying the HU and IHF proteins from E.coli, which bind to the minor groove of DNA through two flexible b-strand regions. This type of interaction is of interest since the majority of previously characterized protein-nucleic acid interactions have typically involved direct contact between the protein a-helix and the major groove.  The sequence specificity of the minor groove interaction is examined by monitoring H-bond pairings of nucleotide exocyclic amino and carbonyl groups. The vibrational modes of these exocyclic groups reflect the formation of H-bonds since molecular vibrations are dependent on the masses of the vibrating atoms, the molecular geometry, and the forces that restrain molecules in their equilibrium positions. We are also using fluorescence spectroscopy to probe the binding interaction to gain information regarding the global conformation of the protein-DNA complex. Our experiments focus on utilizing either the natural fluorophores in the protein (e.g. Tyr or Trp residues) or labeling the protein or the DNA with a fluorescent molecule. These fluorescence measurements allow us to probe the conformation of the DNA before and after it binds to the protein and fluorescence resonance energy transfer measurements reveal the relative proximity of the protein to the DNA.

Protein-Protein Interactions. Understanding the forces that govern the interaction of proteins with one another assists in the understanding of such processes as macromolecular assembly, chaperone-assisted protein folding and protein translocation. We study the polymerization of sickle cell hemoglobin as a paradigm for understanding protein-protein interactions. Polymerization of sickle cell hemoglobin results from the one residue mutation (b6 Glu to Val) in the A helix of the protein. This one residue mutation creates a hydrophobic surface that initiates the aggregation of the protein tetramers, by interacting with the b85 Phe and b88 Leu residues on an adjacent tetramer. Our studies are designed to investigate the polymers as they are forming by monitoring the Phe Raman bands, which are reflective of local environment. At present we have established that the tertiary structure of Hb S tetramers differs from that of Hb A in the region of the mutation. This tertiary structural change may have implications for polymer formation. Our current work focuses on a Hb S derivative, which allows us to chemically induce polymerization of the molecule. The mechanism of polymerization is the same for the modified Hb S and native Hb S, as shown by kinetic measurements. Electron micrographs of polymerized Hb demonstrate that the fibers from the modified Hb S are similar in size and shape to those formed by deoxy Hb S. Further work includes a study of Hb S fibers at different stages in the polymerization process and a study of the effect of anti-sickling drugs on Hb S fibers and tetramers.

Recent Publications

The Role of b93 Cys in the inhibition of Hb S Fiber Formation, K. M. Knee, C. K. Roden, M. R. Flory and I. Mukerji (2007) Biophysical Chem., 187:181-193. DOI:10.1016/j.bpc.2007.02.002

Bacterial protein HU dictates the morphology of DNA condensates produced by crowding agents and polyamines, Tumpa Sarker, Iulia Vitoc, Ishita Mukerji, and Nicholas V. Hud.  (2007) Nucleic Acid Research, Vol. 35:951-961 DOI: 10.1093/nar/gkl1093.

Examination of A-Tract Bending using a Fluorescent Adenosine Analogue, K. E. Augustyn, K. Wojtuszewski, M. E. Hawkins, J. R. Knutson and I. Mukerji (2006) Biochemistry, 45:5039-5047

Resonance Raman Spectroscopy, I. Mukerji (August 2005) in Encyclopedia of Life Sciences.  John Wiley and Sons, Ltd: Chichester.  http://www.els.net/ [doi: 10.1038/npg.els.0003113]

Molecular Beacon-Equilibrium Cyclization Detection of DNA-Protein Complexes, Jason Vitko, Iulian Rujan, Lagu Androga, Ishita Mukerji and Philip H. Bolton (2007) . Biophysical J. in revision.

The Role of b93 Cys in Hb S Fiber Formation:  A Spectroscopic Investigation, K. M. Knee, C. K. Roden and I. Mukerji (2005) Biochemistry, submitted.

Effects of HU binding on the equilibrium cyclization of mismatched, curved and normal DNA.  Arthanari, H., Wojtuszewski, K., Mukerji, I., Bolton, PH.  (2004) Biophys. J. 86:1625-31.

The HU-DNA binding interaction probed with UV resonance Raman specroscopy: Structural elements of specificity. Kristi Wojtuszewski and Ishita Mukerji, Protein Science (2004) 13:2416-2428.

HU Binding to Bent DNA: A Fluorescence Resonance Energy Transfer and Antisotropy Study. Kristi Wojtuszewski and Ishita Mukerji, Biochemistry 42 (2003) 3096-3104.

UV Resonance Raman and Circular Dichroism Studies of a DNA Duplex Containing An A₃T₃ Tract:  Evidence for a Premelting Transition and Three-Centered H-bonds, I. Mukerji and A. P. Williams Biochemistry (2002) 41: 69-77.

HU Binding to DNA:  Evidence for Multiple Complexes and DNA Bending, K. Wojtuszewski, M. E. Hawkins, J. L. Cole and I. Mukerji, Biochemistry 40 (2001) 2588-2598.

Lipid and Signal Peptide-Induced Conformational Changes within the C-Domain of Escherichia coli SecA Protein, H. Ding, I. Mukerji and D. Oliver Biochemistry 40 (2001) 1835-1844.

Solubility of Fluormethemoglobin S: Effect of Phosphate and Temperature on Polymerization, M. E. Yohe, K. M. Sheffield and I. Mukerji, Biophys. J. 78 (2000) 3218-3226.

Nucleic Acid Structure Investigated by UV Resonance Raman Spectroscopy:  Protonation Effects and A-Tract Structure, L. Sokolov, K. Wojtuszewski, E. Tsukroff and I. Mukerji, J. Biomolec. Struct. Dyn., Conversation 11, 2 (2000) 327-334.

Nucleotide-binding activity of SecA homodimer is conformationally regulated by temperature and altered by prlD and azi mutations, M. Schmidt, H. Ding, V. Ramamurthy, I. Mukerji and D. Oliver, J. Biol. Chem., 275 (2000) 15440-15448.

Structure of Sickle Cell Hemoglobin Fibers probed with UV Resonance Raman Spectroscopy, L. Sokolov and I. Mukerji, J. Phys. Chem. B. 104 (2000) 10835-10843.

UV Resonance Raman and Circular Dichroism Studies of a DNA Duplex Containing An A3T3 Tract:  Evidence for a Premelting Transition and 3-Centered H-bonds, I. Mukerji and A. P. Williams Biochemistry (2002) Vol. 41, No. 1. 

New Light on Allostery:  Dynamic Resonance Raman Spectroscopy of Hemoglobin Kempsey, X. Hu, K. R. Rodgers, I. Mukerji and T. G. Spiro, Biochemistry 38 (1999) 3462-3467.

Conformational Changes in FmetHbS Probed with UV Resonance Raman and Fluorescence Spectroscopic Methods, L. Sokolov and I. Mukerji,  J. Phys. Chem. 102 (1998) 8314-8319.

A UV Resonance Raman Investigation of Poly(rI):  Evidence for Cation-Dependent Structural Perturbations, I. Mukerji, L. Sokolov and M.-R. Mihailescu, Biopolymers 46 (1998) 475-487.

Temperature Dependent UV Resonance Raman Spectroscopy of the Premelting State of dA-dT DNA, S. S. Chan, R. H. Austin, I. Mukerji and T. G. Spiro, Biophys. J. 72 (1997) 1512-1520.

A UV Resonance Raman Study of Tetrads Formed in Hairpin Dimers of d(A-G)10 at Neutral pH, I. Mukerji, M. C. Shiber, T. G. Spiro and J. R. Fresco, Nucleic Acids Research 24 (1996) 5013-5020.

Nucleic Acid Structure Investigated by UV Resonance Raman Spectroscopy:  Protonation Effects and A-Tract Structure, L. Sokolov, K. Wojtuszewski, E. Tsukroff and I. Mukerji, Journal of Biomolecular Structure and Dynamics: ISSN 0739-1102, Conversation II, Issue #2 (2000) 0-940030-81-0 Proceedings of the Eleventh Conversation, University at Albany, SUNY, June 15-19, 1999. Adenine Press (2000)

Principal Investigator

Ishita Mukerji

Graduate Students

Jonathan King
Anthony Moreno
Iulia Vitoc

Grant Support:

National Sciences Foundation.